This multifaceted project provides new knowledge on the identification and characterization of neurotransmitters, neuromodulators and related enzymes and receptors active at major synapses in brainstem auditory nuclei. The effects of lesions in the cochlea on auditory nerve and cochlear nucleus are determined. Using specific antibodies against GABA and glycine, putative GABAergic and glycinergic neurons in auditory nuclei in guinea pig were identified with immunocytochemistry. In the dorsal cochlear nucleus, double labeling studies showed the presence of neurons containing GABA, neurons containing glycine and neurons containing both GABA and glycine. The coexistence of GABA and glycine immunoreactivities in a population of cell bodies and terminals indicates that both amino acids may be released from these neurons. Projections of glycine- and GABA-containing neurons were determined using immunocytochemistry and retrograde transport of horseradish peroxidase. Monoclonal antibodies against neurofilament subunits were used in immunocytochemical studies of the auditory nerve and spiral ganglion of guinea pig. Type I and Type II spiral ganglion cells differed in the amounts of neurofilament subunits as determined by antibody binding. With experimentally induced hair cell loss, changes in the expressions of neurofilament subunits were apparent in Type 1 spiral ganglion cells. We have continued our characterization of the neurotransmitter, glutamate, by studying its receptors and related enzymes. In collaboration with the Laboratory of Molecular Biology, NINCDS, a rat expression library has been screened with antibodies against glutaminase. A glutaminase cDNA was isolated and characterized. Kainic acid receptors were solubilized from rat and frog brain. The soluble receptors contained high and low affinity components and were pharmacologically similar to the membrane-associated receptors. Ion exchange chromatography was used to purify the frog brain receptor.